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Br J Pharmacol. 2016 December; 173(23): 3386–3401.
Published online 2016 November 1. doi:  10.1111/bph.13634
PMCID: PMC5738664

Functional pharmacological characterization of SER100 in cardiovascular health and disease

Abstract

Background and Purpose

SER100 is a selective nociceptin (NOP) receptor agonist with sodium‐potassium‐sparing aquaretic and anti‐natriuretic activity. This study was designed to characterize the functional cardiovascular pharmacology of SER100 in vitro and in vivo, including experimental models of cardiovascular disease.

Experimental Approach

Haemodynamic, ECG parameters and heart rate variability (HRV) were determined using radiotelemetry in healthy, conscious mice. The haemodynamic and vascular effects of SER100 were also evaluated in two models of cardiovascular disease, spontaneously hypertensive rats (SHR) and murine hypoxia‐induced pulmonary hypertension (PH). To elucidate mechanisms underlying the pharmacology of SER100, acute blood pressure recordings were performed in anaesthetized mice, and the reactivity of rodent aorta and mesenteric arteries in response to electrical‐ and agonist‐stimulation assessed.

Key Results

SER100 caused NOP receptor‐dependent reductions in mean arterial blood pressure and heart rate that were independent of NO. The hypotensive and vasorelaxant actions of SER100 were potentiated in SHR compared with Wistar Kyoto. Moreover, SER100 reduced several indices of disease severity in experimental PH. Analysis of HRV indicated that SER100 decreased the low/high frequency ratio, an indicator of sympatho‐vagal balance, and in electrically stimulated mouse mesenteric arteries SER100 inhibited sympathetic‐induced contractions.

Conclusions and Implications

SER100 exerts a chronic hypotensive and bradycardic effects in rodents, including models of systemic and pulmonary hypertension. SER100 produces its cardiovascular effects, at least in part, by inhibition of cardiac and vascular sympathetic activity. SER100 may represent a novel therapeutic candidate in systemic and pulmonary hypertension.

Abbreviations

AQP
aquaporin
EFS
electrical field stimulation
GIRK
G‐protein‐gated inwardly rectifying potassium channel
HF
high frequency
HR
heart rate
HRV
heart rate variability
LF
low frequency
L‐NAME
L‐NG‐nitroarginine methylester
MABP
mean arterial blood pressure
NOP/ORL‐1 receptor
nociceptin/orphanin FQ peptide receptor
PE
phenylephrine
PH
pulmonary hypertension
SER100
Ac‐RYYRWKKKKKKK‐NH2
SHR
spontaneously hypertensive rat
VLF
very low frequency
WKY
Wistar‐Kyoto rat

Tables of Links

TARGETS
GPCRs a Catalytic receptors d
κ receptor VEGFR‐2
NOP receptor Enzymes e
Voltage‐gated ion channels b Adenylyl cyclase
GIRK (Kir3.x) channels NOS
Other ion channels c
AQP channels

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (a,b,c,d,eAlexander et al., 2015a,b,c,d,e).

Introduction

Nociceptin modulates cardiac, vascular and renal function via actions in the central nervous system and periphery (Calo' et al., 2000). These effects are triggered by activation of a specific nociceptin receptor, NOP (also known as the orphanin FQ receptor or opiate receptor‐like 1, ORL‐1) receptor. The peptide provokes hypotensive and bradycardic effects and also diuresis in anaesthetised and conscious rodents (Chu et al., 1999; Burmeister and Kapusta, 2007), reducing cardiac output and total peripheral resistance (Kapusta, 2000). Indeed, many of the cardiovascular effects of SER100 are absent in NOP KO mice, indicating the pivotal role of these receptors in the pharmacodynamic profile of the compound (Burmeister and Kapusta, 2007; Fischetti et al., 2009b), and antagonism of the NOP receptor in sepsis reduces microvascular leak and leukocyte extravasation (Brookes et al., 2013). The NOP receptor is Gi/o‐coupled and, as a result, has been shown to reduce adenylyl cyclase activity, open G‐protein‐gated inwardly rectifying K+ channels, and block voltage‐sensitive Ca2 + channels, at least in neuronal cells and tissues (Knoflach et al., 1996; Vaughan and Christie, 1996; Hawes et al., 2000); these effects combine to produce a general dampening of neuronal excitability and neurotransmitter release. However, the mechanisms underpinning the cardiovascular actions of NOP receptor activation remain incompletely understood. One potential mechanism explaining the depressant cardiovascular effects evoked by nociceptin is via inhibition of sympathetic activity with concomitant increases in parasympathetic drive (Giuliani et al., 1997). Alternatively, stimulation of endothelial NO synthesis has been implicated (Lin et al., 2000), although the vasodilator properties of nociceptin in the rat hindquarters vascular bed are insensitive to the NOS inhibitor, L‐NG‐nitroarginine methylester (L‐NAME) (Champion et al., 2002). Additionally, studies in isolated arteries have shown nociceptin to have a direct (i.e. endothelium‐independent) vasodilator activity (Kapusta, 2000), possibly via the release of histamine (Brookes et al., 2007). Consequently, downstream signalling triggered by activation of the NOP receptor in the cardiovascular system requires further elucidation.

SER100 (previously known as ZP120, Ac‐RYYRWKKKKKKK‐NH2) is a peripherally acting, highly potent and selective NOP receptor partial agonist (Rizzi et al., 2002; Kapusta et al., 2005; Fischetti et al., 2009b). It was developed by coupling a chain of six lysine residues to an existing NOP receptor partial agonist hexapeptide, Ac‐RYYRWK‐NH2 (Dooley et al., 1997) to improve metabolic stability. There are a plethora of studies focusing on the renal activity of SER100 since it possesses sodium and potassium –sparing aquaretic effects, in part via modulation of expression and activity of water channel aquaporins (AQPs) (Kapusta et al., 2005; van Deurs et al., 2009; Hadrup et al., 2004). However, akin to nociceptin the peptide has been reported to exert additional cardiovascular actions. For example, SER100 exerts a vasorelaxant effect on electrically stimulated‐mesenteric arteries in vitro by pre‐junctional inhibition of noradrenaline release (Simonsen et al., 2008), and in vivo acute dosing produces a mild hypotensive response without reflex tachycardia (Kapusta et al., 2005). Beyond this limited number of studies, there is little or no information regarding the pharmacology of SER100 in the cardiovascular system, certainly in the context of cardiovascular disease.

In terms of clinical evaluation, SER100 was originally assessed in a randomized, double‐blind, placebo controlled Phase II trial as add‐on therapy in patients with sub‐acute decompensated chronic heart failure (NCT00283361); however, the clinical development of the peptide for this indication was terminated prematurely due to significant hypotensive activity, primarily on systolic blood pressure (SBP). Nonetheless, as a result of this profound drop in SBP, SER100 (10 mg, s.c., bid) was investigated in a randomized, placebo‐controlled study in patients with treatment‐resistant isolated systolic hypertension (NCT01987284) and found to produce a meaningful and long lasting drop in SBP (~7 mmHg) and diastolic (~4 mmHg) blood pressure (DBP), as well as being safe and well‐tolerated.

The therapeutic interest in SER100, coupled to the lack of mechanistic insight regarding its cardiovascular pharmacodynamic profile, promoted the current study, which was designed to more fully characterize the in vitro and in vivo pharmacology of SER100 in health and disease, including models of both systemic and pulmonary hypertension (PH), and to investigate the underlying pathways.

Methods

All experiments were conducted according to the Animals (Scientific Procedures) Act 1986, United Kingdom and had approval from a local ethics committee within Barts and The London School of Medicine. Animals were housed in a temperature‐controlled environment in a 12‐h light–dark cycle. Food and water were accessible ad libitum. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015).

Isolated thoracic aorta

Male mice (C57B/J7; 9–12 weeks old), Sprague–Dawley (6–8 weeks old), spontaneously hypertensive (SHR; 16–18 weeks) or Wistar‐Kyoto (WKY; 16–18 weeks) rats were killed by cervical dislocation. The thoracic aorta was dissected and rings (~4 mm length) mounted in organ baths containing physiological salt solution (PSS; composition in mM: NaCl 119, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, and glucose 5.5), maintained at 37°C and gassed with 5% CO2 in O2. Changes in isometric tension were measured in the tissues under a basal tension of 0.3 (mice) and 1 (rats) g. After equilibration, vessels were repeatedly contracted with KCl (48 mM) until responses were reproducible. Following this a cumulative concentration–response curves was constructed to the α1‐adrenoceptor agonist phenylephrine (PE; 0.001–30 μM). Arteries were then pre‐contracted with PE (EC80) and, once a stable response was achieved, a concentration–response curve to ACh (0.001–1 μM) was constructed, or a single application of ACh (1 μM) was used to test the presence of endothelium (endothelium intact tissues were defined as having >50% relaxation of PE‐induced tone following addition of ACh; vessels not meeting this criterion were discarded). The vessels were then washed to restore basal tone and contracted again with PE (EC80) and, once a stable response was achieved, cumulative concentration–response curves were constructed to SER100 (0.0001–10 μM) or nociceptin (0.0001–1 μM) in the absence or presence of L‐NAME (300 μM, 30 min) or selective NOP receptor antagonist (BAN‐ORL‐24; 1 μM) (Fischetti et al., 2009a).

Isolated mesenteric artery

Male mice (C57B/J7; 9–12 weeks old), Sprague–Dawley (6–8 weeks old), SHR (16–18 weeks) or WKY (16–18 weeks) rats were killed by cervical dislocation. The mesentery was removed and placed in PSS (composition as above) and third‐order arteries mounted in an automated tension myograph (Danish Myotechnology, Aarhus, Denmark), as previously described (Villar et al., 2007). After an equilibration period of 45 min, vessels were normalized using an automated, step‐wise protocol, and vessel diameter was determined (Mulvany and Halpern, 1977). Following normalization, each vessel was contracted repeatedly with the thromboxane A2‐mimetic 9,11‐dideoxy‐11α,9α‐epoxymethano‐PGF (U46619; 1 μM) or PE (10 μM, WKY and SHR) until the response was reproducible. Vessels were then precontracted with a submaximal concentration of U46619 (EC50) or PE (EC50; WKY and SHR), and the presence of endothelium was tested by a single application of ACh (1 μM) or relaxation concentration–response curves constructed to ACh (0.001–1 μM; WKY and SHR). Subsequently, vessels were washed thoroughly, pre‐contracted with U46619 (EC50) or PE (EC50; WKY and SHR) and, once a stable response was achieved, cumulative concentration–response curves constructed to SER100 (0.0001–10 μM) or nociceptin (0.0001–1 μM) in the absence or presence of L‐NAME (300 μM, 30 min) or BAN ORL‐24 (1 μM).

Electrical field stimulation

Mesenteric arteries were isolated and set‐up for isometric tension recording as described above but additionally subjected to transmural nerve stimulation using platinum electrodes (Danish Myotechnology) secured within the plastic (non‐conducting) mounting jaws. Electrical field stimulation (EFS; 0.1 ms pulse, 10 V, 16 Hz, 0.01 trains s‐1, 1 s train duration) was applied using an electrical stimulator (Grass S88) until stabilization of the induced contractions. To investigate the effect of SER100 on neurogenic contractions and the dependence of this effect on NOP receptors, increasing cumulative concentrations (1 nM–30 μM) of SER100 were added to the tissue, in the absence and presence of BAN‐ORL‐24 (1 μM). Modulation of the contractile response to EFS by SER100 was investigated by construction of frequency‐response curves (0.1 ms pulse, 10 V, 1–64 Hz, 0.01 trains s‐1, 1 s train duration) in the presence and absence of SER100 (1 μM) in the same tissue. To confirm that responses to EFS were of neuronal (i.e. sympathetic) origin, parallel experiments were undertaken to confirm that EFS‐induced contractions were abolished after treatment with guanethidine (10 μM; data not shown).

Systemic blood pressure and heart rate measurements in anaesthetized rodents

Male endothelial NOS knockout (eNOS KO) mice and wild‐type (WT) littermates (both C57B6 background and 9–12 weeks old), and male SHR or WKY rats (both 16–18 weeks) were anaesthetized with isoflurane (2%; 0.4 L·min−1 oxygen) and placed supine on a thermostatically controlled heating blanket (37°C). The left common carotid artery was isolated and cannulated using a fluid‐filled (heparin; 10 U·mL−1 diluted in 0.9% saline) catheter (AD Instruments) for the measurement of arterial blood pressure with a pressure transducer and the right jugular vein for drug administration (i.v.). When blood pressure and heart rate (HR) had stabilized, bolus doses of SER100 (0.1 mg·kg−1) or nociceptin (0.1 mg·kg−1), or cumulative doses of SER100 (0.0001‐1 mg·kg−1), were administered i.v. in the absence or presence of BAN‐ORL‐24 (10 mg·kg−1; 15 min pretreatment) or L‐NAME (100 mg·kg−1·day−1; 3 days; in drinking water). In a separate series of experiments, WT mice were administered the α‐adrenoceptor antagonist prazosin (1 mg·kg−1; i.p.) to investigate the effect of SER100 on peripheral sympathetic tone. Prazosin treatment caused a fall in MABP of 26.2 ± 2.3 mmHg (confirming effective α‐adrenoceptor blockade), following which an infusion of U46619 (45–60 ng·min−1; i.v.; Model A‐99 syringe pump, Razel Scientific Instruments, Sandown, UK) was administered to return the MABP to baseline before constructing a dose–response curve to bolus addition of SER100 (0.0001‐1 mg·kg−1). MABP and HR were recorded continuously and measured using a PowerLab data acquisition systems and Chart 5 software (ADInstruments Ltd, Oxfordshire, UK). Responses were calculated as the maximum change ([big up triangle, open]) in both parameters. In SHR and WKY, studies investigating the maximum change in MABP, results were normalized to the baseline BP since the resting BP was significantly greater in SHR.

Radiotelemetric recording in vivo

Radiotelemetric transmitters (HD‐X11, Data Sciences International) were implanted in the aortic arch, via the left carotid artery, of male C57B6J mice (9–12 weeks‐old) anaesthetized with 2% isoflurane (0.4 L·min−1 oxygen) and recording leads tunnelled s.c in a conventional lead II ECG configuration using aseptic surgical technique. After 10 days recovery, MAP, HR and surface ECG parameters (e.g. RR, PR, QRS, and QTc intervals and P duration) were recorded in conscious mice for 24 h, then ALZET osmotic minipumps (Model 1002; Alzet, California, USA) filled either with saline (vehicle) or SER100 (1 mg·kg−1 ·day−1) were implanted s.c for 14 days. During recording periods, mice were left undisturbed and maintained on a 12 h light/dark cycle (0700–1900 h). MAP, HR and surface ECG parameters were acquired at 0, 3, 7, 10 and 14 days for 2 min every 15 min, and mean values were calculated for every time point (Dataquest Art Acquisition System). Three ECG traces (2 min periods) during periods of inactivity were used for ECG analysis using an ECG extension module of Chart 6.0 software (ADInstruments).

Heart rate variability analysis

Heart rate variability (HRV) was analysed using the HRV extension module of Chart 6.0 (ADInstruments). A threshold sensing algorithm was applied to detect R‐R intervals from the telemetric ECG traces. Artefacts and ectopic beats (defined as those that were two times above or below the average R‐R interval) were excluded from the analysis. Four waveforms (2 min periods) with no erratic fluctuations, during a phase of inactivity were selected from data recorded during the light period. HRV data were analysed in both frequency and time domains using standard HRV parameters (Zuberi et al., 2008). Frequency domain analysis was performed after fast Fourier transform using 1024 spectral points and a half overlap within a Welch window. Cut‐off frequencies previously determined to be accurate for mice (Zuberi et al., 2008) were used to divide signal into three components, very low frequency (VLF <0.4 Hz), low frequency (LF 0.4–1.5 Hz), and high frequency (HF 1.5–4.0 Hz). Normalization to exclude VLF was performed, and spectral variability at each bandwidth was normalized to the total spectral area.

Hypoxia‐induced PH

Male C57B6J mice (8–10 weeks) were randomly assigned to one of five groups as follows (Table 1).

Table 1
Experimental groups with respective treatments

Normoxic animals were maintained at 21% O2 throughout. Hypoxic animals were exposed to 10% O2 in a normobaric chamber for 14 days (to confirm development of a PH phenotype) or 35 days (to investigate reversal of established PH) and administered the VEGF receptor antagonist sugen 5416 (suspension in 0.5% [w.v‐1] carboxymethylcellulose sodium, 0.9% [w.v‐1] sodium chloride, 0.4% [v.v‐1] polysorbate 80, 0.9% [v.v‐1] benzyl alcohol in deionized water; 20 mg·kg−1, s.c., 1× per week for 3 weeks) (Ciuclan et al., 2011). Animals received SER100 (1 mg·kg−1·day−1) or saline delivered by osmotic minipump (model 1004; Alzet, USA) implanted s.c at day 14.

Hemodynamic measurements

Haemodynamic measurements including right ventricular systolic pressure (RVSP) and MABP were recorded as we have described previously (Baliga et al., 2012; Bubb et al., 2014). Briefly, mice were anaesthetized with 1.5% isofluorane, dissolved in O2 and delivered at 0.4 L·min−1, and placed supine on a thermostatically controlled heating blanket (37°C). To measure MABP, the left common carotid artery was isolated and a fluid filled catheter introduced into the vessel. To measure RVSP, the right jugular vein was isolated and a Millar micro‐manometer tipped catheter (Millar MicroTip 1.4F catheter, Millar Instruments, USA) was introduced into the superior vena cava and then advanced into the right ventricle. Both MABP and RVSP were recorded onto a pre‐calibrated PowerLab system (ADInstruments) running Labchart 6.0 software. Following measurement of pulmonary and systemic haemodynamics, animals were killed by anaesthetic overdose and exsanguination, the heart was removed, and heart chamber weights were measured to evaluate right ventricular hypertrophy (RVH; right ventricle to left ventricle plus septum ratio; RV/[LV + S]). In six animals, the left lung lobe was isolated by ligation and removed and snap frozen in liquid N2. The remaining four lung lobes were cannulated via the trachea and fixed by inflation with 4% paraformaldehyde in PBS at physiological pressure before paraffin embedding and sectioning.

Histological evaluation of pulmonary vascular remodelling

Transverse formalin‐fixed lung sections were stained with an anti‐smooth muscle actin (clone 1A4; 1:1000 dilution; Dako, UK) antibody. Pulmonary vascular muscularisation (i.e. remodelling) was determined by counting vessels of less than 100 μm in diameter in each lung section, and defining according to degree of muscularization: fully muscularized (two distinct and continuous elastic lamina), partially muscularized (second elastic lamina not continuous) and non‐muscularized (single elastic lamina). Approximately 75 vessels were counted per section from six animals in each group and the proportion of vessels in each category was expressed as a percentage of total vessels counted.

Immunoblot analysis of AQP‐1 expression in lung homogenates

Mice exposed to normoxia or 5 weeks hypoxia in the absence and presence of SER100 (1 mg·kg−1·day−1) were killed by cervical dislocation, the lungs were extracted, snap frozen in liquid N2, and homogenates were generated using a Precellys Tissue Homegenizer (Precellys, UK). AQP‐1 protein expression was determined by immunoblot using primary anti‐AQP‐1 antibody (Abcam UK; 1:500) and secondary horse‐radish peroxidase conjugated goat anti‐rabbit IgG antibody (Dako, UK 1:2000). Bands were quantified by densitometry using ImageJ and normalized to the loading control (anti‐actin, 1:5000, Millipore, UK; secondary antibody horse‐radish peroxidase conjugated anti‐mouse IgG, Dako, UK).

Effect of SER100 on pulmonary microvascular endothelial cell proliferation

Primary murine pulmonary endothelial cells were isolated as described previously (Khambata et al., 2011). Briefly, mice exposed to normoxia or 5 weeks hypoxia in the absence and presence of SER100 (1 mg·kg−1·day−1) were killed by cervical dislocation, and lungs were extracted and placed into cold DMEM/F12 media. Lungs were minced using scissors and incubated in collagenase (type 1a, 0.1%, Sigma‐Aldrich, UK) for 1 h at 37°C, filtered (70 μm; Millipore, UK) and re‐suspended in endothelial cell growth medium (ECGM) containing DMEM/F12 (Gibco, UK), 20% bovine serum (Gibco, UK), 50 U·mL−1 penicillin and 0.5 mg·mL−1 streptomycin (Sigma‐Aldrich, UK), 50 μg·mL−1 endothelial cell growth supplement (Sigma‐Aldrich UK) and 3 μg·mL−1 endothelial cell growth serum/heparin (Promocell, UK) and were plated in a 75cm2 gelatin‐coated flask (Corning® Biocoat, UK). Cells were grown to ~80% confluence, before undergoing positive selection for endothelial cells using magnetic bead separation (Dynabeads, sheep anti‐rat IgG, 4 × 106 beads.mL‐1, Life Technologies, UK) coated with 50 ng·mL−1 CD31 (rat anti‐mouse, Affymetrix, UK), re‐plated and grown to ~80% confluence before a second positive selection using 5 μg·mL−1 CD102 (rat anti‐mouse, BD Pharmingen, UK). For all experiments, endothelial cells were removed from flasks using trypsin and re‐suspended in appropriate medium.

To assess proliferation, cells were seeded onto 96‐well plates at a density of 1 × 105 cells per well, grown for 24 h, and starved in low serum for 24 h. Cells were then incubated in medium containing 20% FBS in the absence and presence of SER100 (10 nmol·L−1 to 10 μmol·L−1) and counted at 24 h using BrdU incorporation (Roche Diagnostics, UK).

Effect of SER100 on pulmonary microvascular endothelial cell migration

Primary pulmonary microvascular endothelial cells were isolated as above. Endothelial cells were plated in gelatin‐coated wells of a 96 well plate (Corning® Biocoat, UK) at a density of 2.5 × 105 cells per well and grown to confluence. Twelve hours prior to and for the duration of the experiment, cells were incubated in a media containing 1% serum. A scratch was performed using a 10 μL sterile pipette, and images were taken at regular intervals over a 24 h period to monitor scratch closure. Cells were treated with vehicle (DMEM/F12 alone) or SER100 (10 nM to 10 μM, dissolved in DMEM/F12).

Statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). All data are shown as mean ± SEM and for vascular reactivity studies curves were fitted to the data using nonlinear regression. Tests of significance between curves were conducted using two‐way ANOVA with repeated measures. For in vivo studies, changes in haemodynamic, ECG and HRV parameters, were analysed by one‐way ANOVA, with Bonferroni post hoc tests where appropriate. For the PH model, analyses were performed by one‐way ANOVA, with Bonferroni post hoc tests, with the exception of the endothelial cell migration which was analysed by two‐way ANOVA with repeated measures. All statistical analyses were performed using GraphPad Prism version 5. A P value <0.05 was considered to indicate statistical significance. The n values indicate the number of animals in each group, and all data points were included in the statistical analyses.

Reagents

All reagents were obtained from Sigma with the following exceptions: SER100 (Serodus), nociceptin (Phoenix Pharmaceuticals), BAN‐ORL‐24 (Tocris), guanethidine (Cambridge Biosciences) and U46619 (Enzo Life Sciences). All drugs were dissolved and diluted in distilled water (in vitro experiments) and sterile saline (in vivo experiments) except U46619, which was dissolved in ethanol (1:1) and then diluted in saline.

Results

Effect of SER100 and nociceptin on rodent vascular reactivity

In mouse thoracic aorta and mesenteric arteries, SER100 caused a modest concentration‐dependent contraction of pre‐constricted vessels (Figure 1). In sharp contrast, in rat vessels SER100 caused a small concentration‐dependent relaxation (Figure 1). Since SER100 is described as a highly selective and potent NOP partial agonist, we investigated if nociceptin induces a similar response profile to that of SER100 in the rat vasculature. Akin to SER100, nociceptin caused a small concentration‐dependent relaxation in rat mesenteric vessels (Figure 1); however, nociceptin failed to elicit any vasorelaxant activity in the rat aorta at the concentration range employed (Figure 1); it should be noted however that an inability to attain nociceptin concentrations as high as SER100 in this setting (i.e. >1 μM), due to cost, may have masked a small vasorelaxant response.

Figure 1
Effect of SER100 on mouse and rat vascular reactivity. Concentration‐response curves to SER100 (A, B, D and E) or nociceptin (C and F) in mouse mesenteric arteries (A), rat mesenteric arteries (B and C), mouse thoracic aorta (D) or rat thoracic ...

Previous reports have suggested that vasorelaxant responses to nociceptin may be sensitive to blockade by L‐NAME (Lin et al., 2000), although this mechanism remains unclear (Champion et al., 2002). To determine if the modest vasorelaxant responses to SER100 and nociceptin in rat vessels were dependent on the release of NO, parallel studies were conducted in the presence of L‐NAME (300 μM, 30 min). NOS inhibition significantly attenuated the response to SER100 in rat aorta (Figure 1) but was unable to block identical relaxations in rat mesenteric arteries (Figure 1). In contrast, nociceptin responses were significantly reduced in the presence of L‐NAME in mesenteric vessels (Figure 1).

Effect of chronic exposure to SER100 on haemodynamics in conscious mice

To establish whether the modest vascular effects of SER100 observed in vitro were mirrored by the in vivo (cardio)vascular pharmacological profile of the compound, haemodynamic changes in response to SER100 (with nociceptin again used as a positive control) were measured in conscious mice using radiotelemetry. The baseline MABP was 103.8 ± 0.5 mmHg and HR 577.6 ± 2.8 beats.min−1 (Figure 2). Continuous infusion of SER100 (1 mg·kg−1.day−1) caused a significant decrease in MABP and HR immediately following initiation of treatment that remained reduced throughout the 14 day treatment period. In the case of MABP, the effect of SER100 increased temporally, with the maximum depressor effect achieved at day 10 ([big up triangle, open]MABP = −5.8 ± 0.6 mmHg, [big up triangle, open]HR = −36.2 ± 3.8 beats.min−1; Figure 2). The hypotensive action of SER100 was equivalent against SBP ([big up triangle, open]MABP = −6.0 ± 0.6 mmHg) and DBP ([big up triangle, open]MAP = −5.6 ± 0.5 mmHg).

Figure 2
Haemodynamic effect of SER100 in conscious mice. MABP (A), SBP (B), DBP (C), HR (D), changes in HF power (E) and LF/HF ratio (F) in conscious, telemetred mice following infusion of SER100 (1 mg·kg per day) for 3, 7, 10 or 14 days. ...

Effect of SER100 on ECG parameters and heart rate variability (HRV)

SER100 did not alter any of the direct ECG parameters determined (Table 2) but did modulate HRV. Measurements in the time domain showed a modest increase in RR variability following administration of SER100 (1 mg·kg−1.day−1), and this was accompanied by a significant reduction in LF/HF ratio (Figure 2) indicative of an inhibition of sympathetic activity. Moreover, SER100 caused a concomitant, minor increase in the mean HF component, representative of parasympathetic activation (Figure 2).

Table 2
ECG parameters under control (baseline) conditions and following treatment with SER100 over 14 days

Role of NOS and α‐adrenoceptor activity in the depressor responses of SER100 in vivo

In vitro data suggested that in certain vessels, the vasorelaxant activity of SER100 (rat aorta) and nociceptin (rat mesentery) was attenuated in the presence of NOS inhibition, as has been previously reported in the case of NOP activation by nociceptin (Champion et al., 2002). To study whether endothelium‐derived NO was implicated in the hypotensive properties of SER100 in vivo, the compound was administered to WT and eNOS KO mice and haemodynamic measurements were recorded. In eNOS KO mice, the administration of bolus doses of SER100 (0.0001 mg·kg−1; i.v.) caused dose‐dependent decreases in MABP, which were not significantly different from WT mice (Figure 3), indicating that this enzyme is unlikely to be involved in the hypotensive actions of SER100. Since compensatory NO production by alternate NOS isoforms has been hypothesized to contribute to the maintenance of local vascular tone and blood pressure, essentially identical studies were conducted in control animals and mice treated with L‐NAME (100 mg·kg−1.day−1; 3 days; drinking water). However, L‐NAME treatment did not alter the hypotensive effect of SER100 (Figure 3). Indeed, if anything the decrease in MABP in response to SER100 was greater in eNOS KO and L‐NAME–treated mice (Figure 3).

Figure 3
Role of NOS in the haemodynamic responses to SER100 in anaesthetised mice. Changes in [big up triangle, open]MABP in response to bolus doses of SER100 (0.0001‐1 mg·kg−1) in WT and eNOS KO mice (A), WT animals in the absence (Control) ...

In addition, the depressor effect of SER100 was significantly reduced following α‐adrenoceptor blockade with prazosin, substantiating the in vitro observations that the inhibitory effect of SER100 is elicited, at least in part, via dampening of sympathetic input to the resistance vasculature (Figure 3).

Effect of SER100 and nociceptin on vascular reactivity in spontaneously hypertensive rats

Since clinical interest in SER100 is centred on potential utilization in cardiovascular disease, presently in isolated systolic hypertension, we explored the (cardio)vascular pharmacology of the compound in a model of hypertension, as provided by the SHR. As expected, the MABP was higher in SHR (186.3 ± 5.28 mmHg) compared with normotensive WKY (113.6 ± 3.8 mmHg; P < 0.001; n = 5) confirming the development of hypertension in the SHR. Furthermore, in line with previous reports (Konishi and Su, 1983; Sim and Singh, 1987), endothelial dysfunction was observed in both the aorta and mesenteric resistance arteries, as evidenced by a reduction in the vasorelaxant capacity of ACh (relaxation to 1 μM; WKY: 87.5 ± 2.5%, SHR: 23.6 ± 5.7%; P < 0.05; n = 5).

In mesenteric arteries from WKY rats, SER100 caused a modest relaxation that was enhanced in SHR (Figure 4). These responses were not affected in the presence of BAN‐ORL‐24 (Figure 4). In rat aorta, SER100 caused a small relaxation in both strains that was also insensitive to NOP blockade (Figure 4). These observations suggest an increased sensitivity to SER100 in the small mesenteric arteries from animals with a hypertensive phenotype, but that this enhanced vasorelaxant activity is not due to modification of NOP. Nociceptin did not relax pre‐contracted mesenteric arteries or aorta from SHR in contrast to SER100 (Figure 4). This differential activity may, again, be explained by the inability to achieve nociceptin concentrations as high as SER100.

Figure 4
Vascular responses to SER100 and nociceptin in SHR. Concentration‐response curves to SER100 (A, B, D and E) and nociceptin (C and F) in mesenteric arteries (A, B and C) and thoracic aorta (D, E and F) from WKY (A and D) and SHR (B, C, E and F) ...

Effect of SER100 in the spontaneous hypertensive rat (SHR)

Further studies were conducted to investigate whether the increased sensitivity to SER100 in isolated resistance arteries from SHR translated to a more prominent hypotensive effect in vivo. Acute, bolus dosing of SER100 (0.0001–0.01 mg·kg−1) to anaesthetised rats induced a dose‐dependent decrease in MABP and HR that was significantly enhanced in SHR compared with WKY (Figure 5). Interestingly, in both strains the depressor effect of SER100 (0.01 mg·kg−1) was equivalent against SBP and DBP (WKY: [big up triangle, open]SBP = −6.8 ± 1.9 mmHg, [big up triangle, open]DBP = −17.8 ± 11.2 mmHg; SHR: [big up triangle, open]SBP = −32.5 ± 4.8 mmHg, [big up triangle, open]DBP = −46.3 ± 5.8 mmHg; P > 0.05, [big up triangle, open]SBP vs [big up triangle, open]DBP in each strain).

Figure 5
Role of the NOP receptor in hypotensive responses to SER100 in SHR. Depressor effects of SER100 (0.0001–0.01 mg·kg−1) on MABP (A) and HR (B) in WKY and SHR in the absence and presence of the NOP receptor antagonist BAN ...

The hypotensive actions of SER100 were essentially abolished in the presence of the NOP receptor antagonist BAN ORL‐24 (Figure 5), confirming this receptor as the exclusive pathway triggered by SER100 to elicit its cardiovascular activity in vivo. This mirrored the depressor actions of nociceptin on MABP and HR, which were also sensitive to BAN‐ORL‐24 (Figure 5). Of note, BAN‐ORL‐24 did not alter MABP per se, indicating that NOP does not contribute to the dynamic regulation of systemic blood pressure (data not shown).

SER100 inhibits electrical field stimulation (EFS)‐induced contraction

Since the vasoreactivity of SER100 was relatively modest in isolated vessels per se, and the HRV evaluation in vivo intimated an effect on sympathetic drive, we explored if SER100 exerts its vasorelaxant effect by altering sympathetic tone using isolated vessels. Here, SER100 inhibited, in a concentration‐dependent manner, the electrically‐induced contractions of mouse mesenteric arteries, and pretreatment with SER100 (1 μM) significantly attenuated EFS‐induced contraction in the range of frequencies 4 to 16 Hz (Figure 6). Furthermore, the inhibitory effect of SER100 against EFS‐induced contractions was sensitive to BAN‐ORL‐24, indicating NOP activation underlies this pharmacological action (Figure 6). Such findings support an anti‐sympathetic effect of SER100, in vitro and in vivo, which may underpin its vasorelaxant and/or depressor effects, at least in part.

Figure 6
Effect of SER100 on sympathetic activity in vitro. Frequency‐dependent responses to EFS (10 V, 1‐64 Hz, 0.1 ms pulse duration, 0.01 trains.s‐1, 1 s train duration; (A) in the absence (Control) and ...

Effect of SER100 on pulmonary haemodynamics

Having demonstrated a potent hypotensive effect of SER100 on systemic haemodynamics, we proceeded to determine if a similar depressor effect is observed in the pulmonary circulation in the context of pulmonary hypertension (PH); this is particularly pertinent based on the anti‐sympathetic activity of SER100 identified above, since sympathetic drive is also increased in PH (Velez‐Roa et al., 2004; Ishikawa et al., 2009).

Hypoxia induced a significant increase in RVSP at 2 weeks (normoxia: 21.7 ± 0.9 mmHg versus hypoxia: 48.1 ± 1.5 mmHg) and 5 weeks (hypoxia: 51.7 ± 1.3 mmHg) that was significantly reversed in the presence of SER100 (42.8 ± 1.1 mmHg; P < 0.05; Figure 7). SER100 had no significant effect on RVSP under normoxic conditions (18.3 ± 1.2 mmHg), although it did tend to reduce RVSP modestly (Figure 7). Concomitantly, this dose of SER100 did not produce a statistically significant reduction in MABP, either under normoxic or hypoxic conditions (Figure 7), indicating a degree of pulmonary selectivity. It should be noted that the effect of SER100 on MABP in this setting was less marked than that observed in the telemetric monitoring studies ([big up triangle, open]MABP = −3.4 mmHg v [big up triangle, open]MABP = −6.0 mmHg; Figure 2), perhaps the results of these animals being anaesthetised during measurement thereby dampening sympathetic drive.

Figure 7
Effect of SER100 on pulmonary haemodynamics, cardiac morphology and vascular remodelling in experimental PH. RVSP (A), MABP (B), RV/[LV + S] ratio (C), and pulmonary vascular remodelling (muscularisation; D and E) in normoxic (Nx) mice ...

Effect of SER100 on right ventricular hypertrophy

Hypoxia induced a significant increase in RVH as indicated by a greater right ventricle to left ventricle plus septum ratio (RV/[LV + S]; normoxia: 0.26 ± 0.004, 2 weeks hypoxia: 0.38 ± 0.013, 5 weeks hypoxia: 0.45 ± 0.018; Figure 7). SER100 produced a significant reversal of the RVH following 5 weeks hypoxia (RV/[LV + S]: 0.38 ± 0.02; P < 0.05, Figure 7), but did not influence RV size in normoxic mice (Figure 7).

Effect of SER100 on pulmonary vascular remodelling

Normoxic animals had a low percentage of muscularised pulmonary small arteries as expected (Figure 4). Exposure to hypoxia caused pulmonary vascular re‐modelling characteristic of PH with a significantly greater percentage of vessels becoming fully muscularised, with a smaller number of partially muscularised arteries. While SER100 reduced the mean number of partially and fully muscularised vessels, this did not reach statistical significance (Figure 4).

Effect of SER100 on pulmonary microvascular endothelial cell proliferation and migration

Endothelial dysfunction/damage, and loss of NO bioavailability, is well‐established to contribute to the development of PH (Budhiraja et al., 2004; Coggins and Bloch, 2007). In the light of this common pathological mechanism, we evaluated whether SER100 affected endothelial proliferation and migration, thereby potentially restoring endothelialisation and NO production in PH.

SER100 (10 nM–10 μM) produced a concentration‐dependent augmentation of pulmonary microvascular endothelial cell growth from mice exposed to 5 weeks hypoxia (10% O2; Figure 8). SER100, at a concentration evoking maximal proliferation (1 μM), caused an increase in pulmonary microvascular endothelial cell migration from mice exposed to 5 weeks hypoxia (10% O2; Figure 8). Interestingly, in animals treated with SER100 (1 mg·kg−1.day−1) in vivo, the basal migration of pulmonary microvascular endothelial cells was significantly greater than mice exposed to hypoxia in the absence of SER100 (Figure 8) and the maximal shift was greater than in cells treated with SER100 in vitro (38.9 ± 8.0 in vitro vs. 71.3 ± 6.6 in vivo; P < 0.05).

Figure 8
Effect of SER100 on endothelial function and AQP‐1 expression. Proliferation (A) and migration (B and C) of pulmonary microvascular endothelial cells from mice exposed to hypoxia (Hx, 10% O2; 5 weeks) plus sugen (20 mg·kg ...

Effect of SER100 on aquaporin (AQP)‐1 expression in lung homogenates

SER100 produces its aquaretic activity by altering the expression and activity of AQP water channels in the kidney (Hadrup et al., 2004; Kapusta et al., 2005; van Deurs et al., 2009). Interestingly, AQP‐1 has been implicated in the pathogenesis of PH and high altitude pulmonary oedema (Leggett et al., 2012; She et al., 2013; Lai et al., 2014). Thus, we examined whether modulation of AQP‐1 might explain the salutary effect of SER100 observed in the experimental model. AQP‐1 expression tended to be up‐regulated in lungs from hypoxic mice when compared with normoxic controls (Figure 8). Notably, AQP‐1 expression was further and significantly increased in hypoxic animals receiving SER100 (1 mg·kg−1.day−1; Figure 8).

Discussion

Herein, we have investigated the cardiovascular pharmacology of SER100 in rodents and the mechanisms involved in these effects. Our results indicate that SER100 elicits a chronic hypotensive effect (with concomitant bradycardia) via NOP receptor activation, independent of NO signalling, but it has a poor vasorelaxant effect per se in isolated arteries. This pharmacodynamic profile is mediated, at least in part, by inhibition of sympathetic drive properties are largely potentiated in a model of hypertension (SHR).

Acutely, SER100 and nociceptin elicited depressant effects on MAP and HR in anaesthetised mice, in accord with previous studies performed in rats using a similar concentration range (Kapusta et al., 2005). The hypotensive and bradycardic effects of both compounds were sensitive to the highly‐selective NOP receptor antagonist BAN‐ORL‐24 (Gavioli and Calo', 2013), demonstrating that SER100 mediates its cardiovascular actions by activation of NOP receptors in vivo, an observation consistent with published in vitro findings (Rizzi et al., 2002; Fischetti et al., 2009b). Interestingly, in this study, the haemodynamic effects of SER100 were maintained over a 2‐week period, and the vasorelaxant, hypotensive and bradycardic effects of SER100 were potentiated in hypertensive rats. Taken together, these properties indicate that this peptide might represent a good candidate for anti‐hypertensive therapy. Indeed, a recently conducted clinical evaluation of SER100 in patients with isolated systolic hypertension (NCT01987284) suggests the compound is safe and well‐tolerated, and produces a clinically meaningful and long‐lasting drop in both SBP and DBP.

Nociceptin has central and peripheral effects, whereas SER100 elicits NOP receptor‐mediated CNS pro‐nociceptive and motor depressant activity when administrated i.c.v. but not when the peptide is injected i.v., even at very high doses, indicating it does not cross the blood–brain barrier to any great extent (Rizzi et al., 2002; Kapusta et al., 2005). Therefore, it is likely that the cardiovascular effects of SER100 are through a peripheral action. One major factor that regulates local blood flow and systemic blood pressure is NO, and the participation of this signalling pathway in the cardiovascular effects of nociceptin has generated some debate, with contradictory effects of NOS inhibition reported (Armstead, 1999; Lin et al., 2000; Champion et al., 2002). Therefore, we explored if NO plays a role in the vasoactivity of SER100 in vitro and in vivo, using both pharmacological and genetic approaches. Previous studies have showed that nociceptin exhibits direct vasodilator actions on diverse vascular beds (Gumusel et al., 1997). Surprisingly, in this study SER100 and nociceptin only elicited modest vasorelaxant effects and, indeed, in some circumstances contractile activity. For example, in isolated murine mesenteric arteries and aorta, SER100 caused a vasocontraction at the highest concentrations, whereas in isolated rat vessels, SER100 elicited a moderate vasorelaxation. These differences may be due to species and vascular bed variation; regardless, the limited vascular effect exerted by SER100 in vitro is unlikely to be responsible for the markedly hypotensive response observed in vivo. The vasorelaxant responses to SER100 and nociceptin were also differentially affected by L‐NAME, with attenuation of vasorelaxant activity of nociceptin and SER100 in the mesenteric artery and aorta respectively. Notably, however, hypotensive responses to SER100 were essentially identical in eNOS KO animals or WT mice receiving L‐NAME, suggesting that endothelium‐derived NO does not contribute to the acute vasodepressor activity of SER100 in vivo. In agreement with this conclusion, Simonsen et al. demonstrated that the vasorelaxant activity SER100 and nociceptin were not affected by endothelial removal in vitro (Simonsen et al., 2008).

If NOP receptor activation by SER100 is not exerting a direct vasorelaxant effect to lower blood pressure, one plausible alternative is that it affects sympathetic outflow, as has been previously suggested (Simonsen et al., 2008). For example, it has been proposed that nociceptin exerts its cardiovascular actions by a concomitant inhibition of sympathetic cardiovascular tone and activation of parasympathetic activity to the heart (Giuliani et al., 1997). The lack of baroreflex‐induced increase in HR in response to the hypotensive activity of SER100 described in this study suggests that this compound might have an effect on sympathetic nerve activity. In fact, several reports have shown that SER100 has inhibitory effects on electrically stimulated mouse vas deferens and rat arteries (Rizzi et al., 2002; Simonsen et al., 2008; Fischetti et al., 2009b). Our data demonstrating inhibition of EFS‐induced contractions of mouse mesenteric arteries by SER100 are in agreement with these findings. We additionally examined this phenomenon in vivo. HRV is widely used as an index of autonomic nervous activity (Laude et al., 2008; Thireau et al., 2008). It is generally accepted that the high‐frequency component represents vagal activity while LF/HF ratio can assess the state of the sympathetic input (Thireau et al., 2008; Nunn et al., 2013). In this study, spectral analysis of HRV revealed a decreased sympathetic activity in conscious mice after chronic administration of SER100 determined by a reduction in the LF/HF ratio. Inhibition of sympathetic activity peaked at 14 days of treatment, which matched reasonably well, from a temporal perspective, to the greatest reduction in MABP (and HR), which reached a maximum at 10–14 days. However, reductions in MABP and HR were observed much more acutely after initiation of SER100 treatment, so additional mechanisms above and beyond effects on sympathetic drive are likely to contribute. In addition, that the depressor response to SER100 is lost in in vivo in the presence of α‐adrenoceptor blockade also supports a sympatholytic‐based mechanism for the vasoactive pharmacology of this compound. Further still, the greater relaxant responses to SER100 in isolated mesenteric vessels from SHR fits with the greater noradrenaline content and release in the arteries from these animals (Gradin et al., 2006). These observations dovetail well with the greater depressor activity of SER100 in vivo in a model of hypertension (SHR). One might argue that the much higher starting pressure in the SHR accounts for such a difference, but this cannot underlie the increased response in its entirety since eNOS KO mice and animals treated with the NOS inhibitor L‐NAME, both have substantially raised MABP, but did not show such an exaggerated response compared to WT/naïve counterparts. These observations suggest that a fundamental mechanistic difference between hypertensive and normotensive states, potentially increased sympathetic activity, underpins the increased potency of SER100. Such a profile would bode well for development of the compound as an anti‐hypertensive agent.

This study also provides evidence of the therapeutic potential of SER100 for PH. Subcutaneous infusion of SER100 decreased RVSP by almost 30% in a preclinical mouse model that was associated with an even greater reduction in RVH. Although not able to reverse RVH completely over the timeframe of this study, SER100 produced an impressive cessation of RVH such that no further increase was observed once SER100 treatment commenced. Further studies are required to examine this effect more closely and elucidate whether SER100 directly affects the myocardium or whether this is secondary to the effects on pulmonary haemodynamics. Interestingly, at the doses employed in this study, SER100 produced a pulmonary‐selective vasodilatory activity. This matches observations in the systemic vasculature in which hypertensive animals (i.e. SHR) exhibited a much greater response to SER100 in terms of hypotension. Thus, common mechanisms, perhaps enhanced sympathetic drive, in the systemic and pulmonary vasculature might underlie the heightened depressor potency of SER100 in hypertensive disorders. Despite these striking beneficial actions on pulmonary haemodynamics and RV morphology, the effects of SER100 against pulmonary vascular remodelling were much more modest, and somewhat disappointing in light of the haemodynamic and cardiac changes.

Endothelial dysfunction is a major component of PH (Budhiraja et al., 2004; Coggins and Bloch, 2007), and we observed substantially increased endothelial cell proliferation and migration after SER100 treatment in this model, which was particularly evident in the cells isolated from mice administered SER100 in vivo. PH is considered an angioproliferative disorder underpinned by the development of apoptotic‐resistant, proliferative endothelial cells that emerge after initial endothelial cell apoptosis; this results in luminal obliteration and the formation of plexiform lesions (Tuder and Voelkel, 2002; Sakao et al., 2009). Therefore, increased endothelial cell survival signalling (e.g. proliferation and migration) may be beneficial in PH. Indeed, monocrotaline‐induced PH is attenuated by over‐expression of VEGF (Campbell et al., 2001) and angiopoietin‐1 (Zhao et al., 2003), both of which are essential for growth and survival of endothelial cells, and are associated with increased endothelial cell migration and proliferation (Lamalice et al., 2007). Moreover, κ‐opioid agonists attenuate hypoxia‐induced PH as a result of improved endothelial function, illustrated by higher eNOS expression and enhanced NO bioavailability (Wu et al., 2013). Together, these data hint that SER100 may be of benefit in PH by supporting endothelial survival and function. This is despite the fact that acute responses to SER100 in the systemic circulation are NO‐dependent.

SER100 was originally developed as an aquaretic drug, decreasing expression of aquaporins, predominantly AQP‐2, in the collecting duct to diminish water reabsorption in the kidney (Hadrup et al., 2004; van Deurs et al., 2009). This mechanism is also responsible for the beneficial effects of SER100 in preclinical models of congestive heart failure, which is associated with increased AQP‐2 expression (Nielsen et al., 1997; Xu et al., 1997; Hadrup et al., 2004). Thus, we speculated that lung water channels may be involved in the salutary effects of SER100 in PH. Since AQP‐1 is the only water channel expressed in pulmonary endothelial cells (Verkman et al., 2000), we investigated the role of this channel in PH. We found a tendency for mice with hypoxia/sugen‐induced PH to have increased expression of AQP‐1 in their lungs, and this was significantly augmented by SER100 treatment (in contrast to the effects of SER100 on renal AQP‐2 expression reported previously). While this observation does not establish a causal relationship between AQP‐1 expression and disease severity in PH, it might suggest that endogenous AQP‐1 expression is increased during PH as an innate defence mechanism and that SER100 promotes this process to bring about a therapeutic effect, at least in part. This hypothesis is supported by previous work revealing that chronic up‐regulation of AQP‐1 promotes endothelial function by facilitating NO movement across cell membranes (Herrera et al., 2006) and augmenting NO‐dependent vasorelaxation (Herrera and Garvin, 2007). Yet, AQP‐1 expression on pulmonary vascular smooth muscle cells promotes migration and proliferation, hallmarks of PH (Leggett et al., 2012; Lai et al., 2014). The overall salutary effect of SER100 therefore suggests the positive influence on endothelial function likely outweighs any negative aspects related to vascular smooth muscle, which may result from a predominant endothelial expression of AQP‐1 in the lung (Verkman et al., 2000).

In conclusion, we have demonstrated a NOP receptor‐dependent, chronic depressor effect of SER100 on BP and HR in vivo. The mechanism responsible for these haemodynamic changes may be, at least in part, inhibition of sympathetic drive. The blood pressure lowering effects of SER100 are potentiated in hypertension, a profile that appears to be matched by data from clinical evaluation of the compound. This exacerbated response to SER100 in hypertension is mirrored by its pharmacology in the pulmonary vasculature, in which SER100 reduced the RVSP and RVH associated with PH. In summary, these observations intimate that this peptide might be a novel therapeutic option for hypertension in both the systemic and pulmonary circulation.

Author contributions

I.C.V., K.J.B. and A.J.M. performed the research. I.C.V., K.J.B., A.J.M., E.S., T.G., F.O.L. and A.J.H. designed the research study. T.G. and E.S. contributed essential reagents. I.C.V., K.J.B., A.J.M. and A.J.H. analysed the data. I.C.V., K.J.B., A.J.M., E.S., T.G., F.O.L. and A.J.H. wrote the paper.

Conflict of interest

This work was supported in part by Serodus AG (in conjunction with the Norwegian Research Council), which holds patents pertaining to the structure and clinical use of SER100.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgments

This work was supported by an Innovations Project Grant from The Norwegian Research Council in conjunction with Serodus ASA.

Notes

Villar I. C., Bubb K. J., Moyes A. J., Steiness E., Gulbrandsen T., Levy F. O., and Hobbs A. J. (2016) Functional pharmacological characterization of SER100 in cardiovascular health and disease. British Journal of Pharmacology, 173: 3386–3401. doi: 10.1111/bph.13634.

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